Rapid classification of biological components

ABSTRACT

A method is disclosed for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an illustrative embodiment of the invention, the analyte is a drug, such as marijuana, cocaine, methamphetamine, methyltestosterone, or mesterolone. The method involves attaching antigens to the surface of a solid support in a preselected pattern to form an array wherein the locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to antigens in the array, thereby forming immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, thereby forming an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject&#39;s identity.

RELATED APPLICATION

This application is a divisional of pending U.S. patent application Ser.No. 10/017,577, filed on Dec. 14, 2001, which claims priority to U.S.Patent Application Ser. No. 60/290,256, filed May 10, 2001, both ofwhich are incorporated by reference herein.

CONTRACTUAL ORIGIN OF THE INVENTION

The United States Government has rights in the following inventionpursuant to Contract No. DE-AC07-94ID13223, Contract No.DE-AC07-99ID13727, and DE-AC07-05ID14517 between the United StatesDepartment of Energy and Battelle Energy Alliance, LLC.

BACKGROUND OF THE INVENTION

This invention relates to assaying biological samples. Moreparticularly, the invention relates to methods for analyzing biologicalsamples comprising antibody profiling. In an illustrative embodiment ofthe invention, the analyzing of biological samples comprises acombination of antibody profiling for characterizing individual specificantibodies in the biological samples and simultaneous assay of ananalyte in the biological samples.

Many methods are known for identifying individuals or biological samplesobtained from such individuals. For example, blood typing is based onthe existence of antigens on the surface of red blood cells. The ABOsystem relates to four different conditions with respect to twoantigens, A and B. Type A individuals exhibit the A antigen; Type Bindividuals exhibit the B antigen; Type AB individuals exhibit both theA and B antigens; and Type O individuals exhibit neither the A nor the Bantigen. By analyzing a sample of a person's blood, it is possible toclassify the blood as belonging to one of these blood groups. While thismethod may be used to identify one individual out of a small group ofindividuals, the method is limited when the group of individuals islarger because no distinction is made between persons of the same bloodgroup. For example, the distribution of the ABO blood groups in the U.S.is approximately 45% O, 42% A, 10% B, and 3% AB. Tests based on otherblood group antigens or isozymes present in body fluids suffer from thesame disadvantages as the ABO blood typing tests. These methods canexclude certain individuals, but cannot differentiate between members ofthe same blood group.

A variety of immunological and biochemical tests based on genetics areroutinely used in paternity testing, as well as for determining thecompatibility of donors and recipients involved in transplant ortransfusion procedures, and also sometimes as an aid in theidentification of humans and animals. For example, serological testingof proteins encoded by the human leukocyte antigen (HLA) gene locus iswell known. Although a good deal of information is known concerning thegenetic makeup of the HLA locus, there are many drawbacks to using HLAserological typing for identifying individuals in a large group. Each ofthe HLA antigens must be tested for in a separate assay, and many suchantigens must be assayed to identify an individual, an arduous processwhen identifying one individual in a large group.

In the past decade, DNA-based analysis techniques, such as restrictionfragment length polymorphisms (RFLPs) and polymerase chain reaction(PCR) have rapidly gained acceptance in forensic and paternity analysesfor matching biological samples to an individual. RFLP techniques areproblematic, however, due to the need for relatively large sample sizes,specialized equipment, highly skilled technicians, and lengthy analysistimes. For forensic applications there is often not enough sampleavailable for this type of assay, and in remote areas the necessaryequipment is often not available. In addition, this technique can takefrom two to six weeks for completion and can result in costly delays ina criminal investigation. Moreover, the cost of RFLP analysis can beprohibitory if screening of many samples is necessary. PCR techniqueshave the advantages over RFLP analysis of requiring much smaller samplesizes and permitting more rapid analysis, but they still requirespecialized equipment and skilled technicians, and they are alsoexpensive.

U.S. Pat. No. 4,880,750 and U.S. Pat. No. 5,270,167 disclose “antibodyprofiling” or “AbP” as a method that overcomes many of the disadvantagesassociated with DNA analysis. Antibody profiling is based on thediscovery that every individual has a unique set of antibodies presentin his or her bodily fluids. R. M. Bernstein et al., Cellular Proteinand RNA Antigens in Autoimmune Disease, 2 Mol. Biol. Med. 105-120(1984). These antibodies, termed “individual-specific antibodies” or“ISAs,” have been found in blood, serum, saliva, urine, semen,perspiration, tears, and body tissues. A. M. Francoeur, AntibodyFingerprinting: A Novel Method for Identifying Individual People andAnimals, 6 Bio/technology 821-825 (1988). ISAs are not associated withdisease and are thought to be directed against cellular components ofthe body. Every person is born with an antibody profile that matches themother's antibody profile. T. F. Unger & A. Strauss, Individual-specificAntibody Profiles as a Means of Newborn Infant Identification, 15 J.Perinatology 152-155 (1995). The child's antibody profile graduallychanges, however, until a stable unique pattern is obtained by about twoyears of age. It has been shown that even genetically identicalindividuals have different antibody profiles. An individual's profile isapparently stable for life and is not affected by short-term illnesses.A. M. Francoeur, supra. Few studies have been conducted on individualswith long-term diseases. Preliminary results, however, indicate that,although a few extra bands may appear, the overall pattern remainsintact. This technique has been used in the medical field to trackpatient samples and avoid sample mix-ups. In addition, the technique hasbeen used in hospitals in cases where switching of infants or abductionhas been alleged. The method has a number of advantages over DNAtechniques, including low cost, rapid analysis (2 hours from the timethe sample is obtained), and simplicity (no special equipment ortraining is necessary). In addition, this method will potentially workon samples that contain no DNA.

WO 97/29206 discloses a method for identifying the source of abiological sample used for diagnostic testing by linking diagnostic testresults to an antibody profile of the biological sample. By generatingan antibody profile of each biological sample, the origin of thebiological sample is identified.

Many assays are now available that use the attachment of specificnucleic acid probes or other biological molecules to surfaces such asglass, silicon, polymethacrylate, polymeric filters, microspheres,resins, and the like. In a configuration where the surface is planar,these assays are sometimes referred to as “biochips.” Initially,biochips contained nucleic acid probes attached to glass or siliconsubstrates in microarrays. These DNA chips are made by microfabricationtechnologies initially developed for use in computer chip manufacturing.Leading DNA chip technologies include an in situ photochemical synthesisapproach, P. S. Fodor, 277 Science 393-395 (1997); U.S. Pat. No.5,445,934; an electrochemical positioning approach, U.S. Pat. No.5,605,662; depositing gene probes on the chip using a sprayer thatresembles an ink-jet printer; and the use of gels in a solution-basedprocess. Arrays of other types of molecules, such as peptides, have beenfabricated on biochips, e.g., U.S. Pat. No. 5,445,934.

While the known methods for using antibody profiling are generallysuitable for their limited purposes, they possess certain inherentdeficiencies that detract from their overall utility in analyzing,characterizing, and identifying biological samples. For example, theknown methods rely on fractionation of antigens by electrophoresis andthen transfer of the fractionated antigens to a membrane. Due todifferences in conditions from one fractionation procedure to another,there are lot-to-lot differences in the positions of the antigens on themembrane such that results obtained using membranes from one lot cannotbe compared with results obtained using membranes from another lot.Further, when colorimetric procedures are used for detecting immunecomplexes on the membrane, color determination can be subjective suchthat results may be interpreted differently by different observers.

In view of the foregoing, it will be appreciated that providing a methodfor analyzing biological samples, wherein lot-to-lot differences inreagents and subjectivity do not affect interpretation of results, wouldbe a significant advancement in the art. More particularly, it would beadvantageous to provide a method for analyzing biological samples byantibody profiling in a biochip format such that analysis would beamenable to automation.

BRIEF SUMMARY OF THE INVENTION

An illustrative embodiment of the invention comprises a method foranalyzing biological material including individual-specific antibodies,comprising: forming an array of multiple antigens by attaching themultiple antigens to the surface of a solid support in a preselectedpattern such that the locations of the multiple antigens are known;obtaining a sample of the biological material and contacting the arraywith the sample such that a portion of the individual-specificantibodies contained in the sample reacts with and binds to antigens inthe array, thereby forming immune complexes; washing the solid supportcontaining the immune complexes such that antibodies in the sample thatdo not react with and bind to the antigens in the array are removed; anddetecting the immune complexes and determining the locations thereofsuch that an antibody profile is obtained.

Illustratively, the detecting of the immune complexes comprises treatingthe solid support having immune complexes attached thereto such that thepresence of immune complexes at a location is characterized by a colorchange as compared to the absence of immune complexes at the location.In one illustrative embodiment, the process of detecting the immunecomplexes further comprises monitoring the solid support with solidstate color detection circuitry for comparing the color patterns beforeand after contacting the array with the sample. In another illustrativeembodiment, the process of detecting the immune complexes furthercomprises obtaining a color camera image before and after contacting thearray with the sample and analyzing pixel information obtainedtherefrom. In still another illustrative embodiment of the invention,the solid support is a surface plasmon resonance chip and the detectingof the immune complexes further comprises scanning the surface plasmonresonance chip before and after contacting the array with the sample andcomparing data obtained therefrom. In yet another illustrativeembodiment of the invention, the detecting of immune complexes comprisesobtaining an image using a charge-coupled device to detect the colorchange comprising fluorescence emission.

In yet another illustrative embodiment of the invention, the method isused as a test for use of drugs. Still another illustrative embodimentof the invention involves analysis of an antibody profile obtained froma forensic sample and comparison with an antibody profile obtained froma sample from a criminal suspect or victim of crime.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows illustrative antibody profiles obtained from saliva samplesaccording to the procedure of Example 1.

FIG. 2 shows comparisons of paired saliva and blood antibody profilesfrom five individuals according to the procedure of Example 1.

FIG. 3 shows antibody profiles obtained from saliva samples from asingle individual after contamination with various adulterants accordingto the procedure of Example 1.

FIG. 4 shows illustrative results obtained from immunoassay of cocainein saliva samples according to the procedure of Example 1.

FIG. 5 shows illustrative results obtained from immunoassay ofmethamphetamine in saliva samples according to the procedure of Example1.

FIG. 6 shows illustrative results of immunodetection of cocaine on aPVDF membrane: strip 5, 0 μg/ml cocaine; strip 6, 0.1 μg/ml cocaine;strip 7, 10 μg/ml cocaine; strip 8, 1000 μg/ml cocaine.

FIG. 7 shows illustrative results of immunodetection of methamphetamineon a PVDF membrane: strip 1, 0 μg/ml methamphetamine; strip 2, 0.1 μg/mlmethamphetamine; strip 3, 10 μg/ml methamphetamine; strip 4, 1000 μg/mlmethamphetamine.

FIG. 8 shows antibody profiles from three different individuals; onestrip of each pair contains no drugs, and the other strip of each paircontains 1000 μg/ml of cocaine and of methamphetamine.

DETAILED DESCRIPTION

Before the present methods for analyzing biological samples aredisclosed and described, it is to be understood that this invention isnot limited to the particular configurations, process steps, andmaterials disclosed herein as such configurations, process steps, andmaterials may vary somewhat. It is also to be understood that theterminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting since thescope of the present invention will be limited only by the appendedclaims and equivalents thereof.

The publications and other reference materials referred to herein todescribe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference. Thereferences discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to a method for analyzing a biological sample from “an animal”includes reference to two or more of such animals, reference to “a solidsupport” includes reference to one or more of such solid supports, andreference to “an array” includes reference to two or more of sucharrays.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

As used herein, “comprising,” “including,” “containing,” “characterizedby,” and grammatical equivalents thereof are inclusive or open-endedterms that do not exclude additional, unrecited elements or methodsteps. “Comprising” is to be interpreted as including the morerestrictive terms “consisting of” and “consisting essentially of.”

As used herein, “consisting of” and grammatical equivalents thereofexclude any element, step, or ingredient not specified in the claim.

As used herein, “consisting essentially of” and grammatical equivalentsthereof limit the scope of a claim to the specified materials or stepsand those that do not materially affect the basic and novelcharacteristic or characteristics of the claimed invention.

As used herein, “solid support” means a generally or substantiallyplanar substrate onto which an array of antigens is disposed. A solidsupport can be composed of any material suitable for carrying the array.Materials used to construct these solid supports need to meet severalrequirements, such as (1) the presence of surface groups that can beeasily derivatized, (2) inertness to reagents used in the assay, (3)stability over time, and (4) compatibility with biological samples. Forexample, suitable materials include glass, silicon, silicon dioxide(i.e., silica), plastics, polymers, hydrophilic inorganic supports, andceramic materials. Illustrative plastics and polymers includepoly(tetrafluoroethylene), poly(vinylidenedifluoride), polystyrene,polycarbonate, polymethacrylate, and combinations thereof. Illustrativehydrophilic inorganic supports include alumina, zirconia, titania, andnickel oxide. An example of a glass substrate would be a microscopeslide. Silicon wafers used to make computer chips have also been used tomake biochips. E.g., U.S. Pat. No. 5,605,662.

As used herein, “array” means an arrangement of locations on the solidsupport. The locations will generally be arranged in two-dimensionalarrays, but other formats are possible. The number of locations canrange from several to at least hundreds of thousands. The array patternand spot density can vary. For example, using a commercially availableGMS 417 Arrayer from Genetic Microsystems (Woburn, Mass.) the spot sizeand density can be selected by the user. With spots of 150 μm diameterand 300 μm center-to-center spacing, more than 1000 spots can be placedin a square centimeter and more than 10,000 spots can be placed on astandard microscope slide. With 200 μm center-to-center spacing, thesenumbers increase to 2500 per square centimeter and more than 25,000 perslide.

As used herein, “colorigenic” refers to a substrate that produces acolored product upon digestion with an appropriate enzyme. Such coloredproducts include fluorescent and luminescent products.

A first step in the present method is to prepare an array of antigens byattaching the antigens to the surface of the solid support in apreselected pattern such that the locations of antigens in the array areknown. As used herein, an antigen is a substance that is bound by anantibody. Antigens can include proteins, carbohydrates, nucleic acids,hormones, drugs, receptors, tumor markers, and the like, and mixturesthereof. An antigen can also be a group of antigens, such as aparticular fraction of proteins eluted from a size exclusionchromatography column. Still further, an antigen can also be identifiedas a designated clone from an expression library or a random epitopelibrary.

In one illustrative embodiment of the invention, antigens are isolatedfrom HeLa cells as generally described in A.-M. Francoeur et al., 136 J.Immunol. 1648 (1986). Briefly, HeLa cells are grown in standard mediumunder standard tissue culture conditions. Confluent HeLa cell culturesare then rinsed, preferably with phosphate-buffered saline (PBS), lysedwith detergent, and centrifuged to remove insoluble cellular debris. Thesupemate contains approximately 10,000 immunologically distinct antigenssuitable for generating an array.

There is no requirement that the antigens used to generate the array beknown. All that is required is that the source of the antigens beconsistent such that a reproducible array can be generated. For example,the HeLa cell supemate containing the antigens can be fractionated on asize exclusion column, electrophoretic gel, density gradient, or thelike, as is well known in the art. Fractions are collected, and eachfraction collected could represent a unique set of antigens for thepurpose of generating the array. Thus, even though the antigens areunknown, a reproducible array can be generated if the HeLa cell antigensare isolated and fractionated using the same method and conditions.

Other methods, such as preparation of random peptide libraries orepitope libraries are well known in the art and may be used toreproducibly produce antigens. E.g., J. K. Scott & G. P. Smith,Searching for Peptide Ligands with an Epitope Library, 249 Science 386(1990); J. J. Devlin et al., Random Peptide Libraries: A Source ofSpecific Protein Binding Molecules, 249 Science 404-406 (1990); S. E.Cwirla et al., Peptides on Phage: A Vast Library of Peptides forIdentifying Ligands, 87 Proc. Nat'l Acad. Sci. USA 6378-6382 (1990); K.S. Lam et al., A New Type of Synthetic Peptide Library for IdentifyingLigand-binding Activity, 354 Nature 82-84 (1991); S. Cabilly,Combinatorial Peptide Library Protocols (Humana Press, 304 pp, 1997);U.S. Pat. No. 5,885,780. Such libraries can be constructed by ligatingsynthetic oligonucleotides into an appropriate fusion phage. Fusionphages are filamentous bacteriophage vectors in which foreign sequencesare cloned into phage gene III and displayed as part of the gene IIIprotein (pIII) at one tip of the virion. Each phage encodes a singlerandom sequence and expresses it as a fusion complex with pIII, a minorcoat protein present at about five molecules per phage. For example, inthe fusion phage techniques of J. K. Scott & G. P. Smith, supra, alibrary was constructed of phage containing a variable cassette of sixamino acid residues. The hexapeptide modules fused to bacteriophageproteins provided a library for the screening methodology that canexamine >10¹² phages (or about 10⁸-10¹⁰ different clones) at one time,each with a test sequence on the virion surface. The library obtainedwas used to screen monoclonal antibodies specific for particularhexapeptide sequences. The fusion phage system has also been used byother groups, and libraries containing longer peptide inserts have beenconstructed. Fusion phage prepared according to this methodology can beselected randomly or non-randomly for inclusion in the array ofantigens. The fusion phages selected for inclusion in the array can bepropagated by standard methods to result in what is virtually an endlesssupply of the selected antigens.

Other methods for producing antigens are also known in the art. Forexample, expression libraries can be prepared by random cloning of DNAfragments or cDNA into an expression vector. E.g., R. A. Young & R. W.Davis, Yeast RNA Polymerase II Genes: Isolation with Antibody Probes,222 Science 778-782 (1983); G. M. Santangelo et al., Cloning of OpenReading Frames and Promoters from the Saccharomyces cerevisiae Genome:Construction of Genomic Libraries of Random Small Fragments, 46 Gene181-186 (1986). Expression vectors that could be used for making suchlibraries are commercially available from a variety of sources. Forexample, random fragments of HeLa cell DNA or cDNA can be cloned into anexpression vector, and then clones expressing HeLa cell proteins can beselected. These clones can then be propagated by methods well known inthe art. The expressed proteins are then isolated or purified and can beused in the making of the array.

Alternatively, antigens can be synthesized using recombinant DNAtechnology well known in the art. Genes that code for many viral,bacterial, and mammalian proteins have been cloned, and thus largequantities of highly pure proteins can be synthesized quickly andinexpensively. For example, the genes that code for many eukaryotic andmammalian membrane-bound receptors, growth factors, cell adhesionmolecules, and regulatory proteins have been cloned and are useful asantigens. Many proteins produced by such recombinant techniques, such astransforming growth factor, acidic and basic fibroblast growth factors,interferon, insulin-like growth factor, and various interleukins fromdifferent species, are commercially available.

In most instances, the entire polypeptide need not be used as anantigen. For example, any size or portion of the polypeptide thatcontains at least one epitope, i.e. antigenic determinant or portion ofan antigen that specifically interacts with an antibody, will sufficefor use in the array.

The antigens, whether selected randomly or non-randomly, are disposed onthe solid support to result in the array. The pattern of the antigens onthe solid support should be reproducible. That is, the location andidentity of each antigen on the solid support should be known. Forexample, in a 10×10 array one skilled in the art might place antigens1-100 in locations 1-100, respectively, of the array.

The proteins may placed in arrays on the surface of the solid supportusing a pipetting device or a machine or device configured for placingliquid samples on a solid support, for example, using a commerciallyavailable microarrayer, such as those from Cartesian Technologies, Inc.(Irvine, Calif.); Gene Machines (San Carlos, Calif.); GeneticMicroSystems (Woburn, Mass.); GenePack DNA (Cambridge, UK); Genetix Ltd.(Christchurch, Dorset, UK); and Packard Instrument Company (Meriden,Conn.).

Relevant methods to array a series of protein antigens onto a surfaceinclude non contact drop on demand dispensing and inkjet technology.Commercially available instruments are available for both methods.Cartesian technologies offers several nanoliter dispensing instrumentsthat can dispense liquid volumes from 20 nL up to 250 μL from 96, 384,1536, 3456, and 9600 well microtiter plates and place them precisely ona surface with densities up to 400 spots/cm². The instruments will spotonto surfaces in a variety of patterns. As the name implies, inkjettechnology utilizes the same principles as those used in inkjetprinters. Microfab Technologies offers a 10 fluid print head that candispense picoliter quantities of liquids onto a surface in a variety ofpatterns. An illustrative pattern for the present application would be asimple array ranging from 10×10 up to 100×100.

There are a number of methods that can be used to attach proteins orother antigens to the surface of a solid support. The simplest of theseis simple adsorption through hydrophobic, ionic, and van der Waalsforces. This method is not optimal, however, since the proteins tend todetach from the surface over time. A preferred attachment chemistryinvolves the use of bifunctional organosilanes. E.g, Thompson andMaragos, 44 J. Agric. Food Chem. 1041-1046 (1996). One end of theorganosilane reacts with exposed —OH groups on the surface of the chipto form a silanol bond. The other end of the organosilane contains agroup that is reactive with various groups on the protein surface suchas —NH₂ and —SH groups. This method of attaching proteins to the chipresults in the formation of a covalent linkage between the protein andthe chip. Other preferred methods that have been used for proteinattachment to surfaces include arylazide, nitrobenzyl, and diazirinephotochemistry methodologies. Exposure of the above chemicals to UVlight causes the formation of reactive groups that can react withproteins to form a covalent bond. The arylazide chemistry forms areactive nitrene group that can insert into C—H bonds, while thediazirine chemistry results in a reactive carbene group. The nitrobenzylchemistry is referred to as caging chemistry whereby the caging groupinactivates a reactive molecule. Exposure to UV light frees the moleculeand makes it available for reaction. Still other methods for attachingproteins to solid supports are well known in the art, e.g., S. S. Wong,Chemistry of Protein Conjugation and Cross-Linking (CRC Press, 340 pp.,1991).

Following attachment of the antigens on the solid support in theselected array, the solid support should be washed by rinsing with anappropriate liquid to remove unbound antigens. Appropriate liquids forwashing include phosphate buffered saline (PBS) and the like, i.e.relatively low ionic strength, biocompatible salt solutions buffered ator near neutrality. Many of such appropriate wash liquids are known inthe art or can be devised by a person skilled in the art without undueexperimentation. E.g., N. E. Good & S. Izawa, Hydrogen Ion Buffers, 24Methods Enzymology 53-68 (1972).

The solid support is then processed for blocking of nonspecific bindingof proteins and other molecules to the solid support. This blocking stepprevents the binding of antigens, antibodies, and the like to the solidsupport wherein such antigens, antibodies, or other molecules are notintended to bind. Blocking reduces the background that might swamp outthe signal, thus increasing the signal-to-noise ratio. The solid supportis blocked by incubating the solid support in a medium that containinert molecules that bind to sites where nonspecific binding mightotherwise occur. Examples of suitable blockers include bovine serumalbumin, human albumin, gelatin, nonfat dry milk, polyvinyl alcohol,Tween 20, and various commercial blockers, such as SEA BLOCK® (trademarkof East Coast Biologics, Inc., Berwick, Me.) and SuperBlock™ (trademarkof Pierce Chemical Co., Rockford, Ill.) blocking buffers.

Following washing for removal of unbound antigens from the array andblocking, the solid support is contacted with a liquid sample to betested. The sample can be from any animal that generates individualspecific antibodies. For example, humans, dogs, cats, mice, horses,cows, and rabbits have all been shown to possess ISAs. The sample can befrom various bodily fluids and solids, including blood, saliva, semen,serum, plasma, urine, amniotic fluid, pleural fluid, cerebrospinalfluid, and mixtures thereof. These samples are obtained according tomethods well known in the art. Depending on the detection method used,it may be required to manipulate the biological sample to attain optimalreaction conditions. For example, the ionic strength or hydrogen ionconcentration or the concentration of the biological sample can beadjusted for optimal immune complex formation, enzymatic catalysis, andthe like.

As described in detail in U.S. Pat. No. 5,270,167 to Francoeur, whenISAs are allowed to react with a set of random antigens, a certainnumber of immune complexes form. For example, using a panel of about1000 unique antigens, about 30 immune complexes between ISAs in abiological sample that has been diluted 20-fold can be detected. If thebiological sample is undiluted, the total number of possible detectableimmune complexes that could form would be greater than 10²³. The totalnumber of possible immune complexes can also be increased by selecting“larger” antigens, i.e. proteins instead of peptides) that have multipleepitopes. Therefore, it will be appreciated that depending on theantigens and number thereof used, the dilution of the biological sample,and the detection method, one skilled in the art can regulate the numberof immune complexes that will form and be detected. The set of uniqueimmune complexes that form and fail to form between the ISAs in thebiological sample and the antigens in the array constitute an antibodyprofile.

Methods for detecting antibody/antigen or immune complexes are wellknown in the art. The present invention can be modified by one skilledin the art to accommodate the various detection methods known in theart. The particular detection method chosen by one skilled in the artdepends on several factors, including the amount of biological sampleavailable, the type of biological sample, the stability of thebiological sample, the stability of the antigen, and the affinitybetween the antibody and antigen. Moreover, as discussed above,depending on the detection methods chosen, it may be required to modifythe biological sample.

While these techniques are well known in the art, examples of a few ofthe detection methods that could be used to practice the presentinvention are briefly described below.

There are many types of immunoassays known in the art. The most commontype of immunoassay is competitive and non-competitive heterogeneousassays, such as enzyme-linked immunosorbent assays (ELISA). In anon-competitive ELISA, unlabeled antigen is bound to a solid support,such as the surface of the biochip. Biological sample is combined withantigens bound to the reaction vessel, and antibodies (primaryantibodies) in the biological sample are allowed to bind to theantigens, thus forming the immune complexes. After the immune complexeshave formed, excess biological sample is removed and the biochip iswashed to remove nonspecifically bound antibodies. The immune complexesare then reacted with an appropriate enzyme-labeled anti-immunoglobulin(secondary antibody). The secondary antibody reacts with antibodies inthe immune complexes, not with other antigens bound to the biochip.Secondary antibodies specific for binding antibodies of differentspecies, including humans, are well known in the art and arecommercially available, such as from Sigma Chemical Co. (St. Louis, Mo.)and Santa Cruz Biotechnology (Santa Cruz, Calif.). After a second washstep, the enzyme substrate is added. The enzyme linked to the secondaryantibody catalyzes a reaction that converts the substrate into aproduct. When excess antigen is present, the amount of product isdirectly proportional to the amount of primary antibodies present in thebiological sample. Preferably, the product is fluorescent orluminescent, which can be measured using technology and equipment wellknown in the art. It is also possible to use reaction schemes thatresult in a colored product, which can be measuredspectrophotometrically, but such calorimetric reactions are notpreferred.

Sandwich or capture assays can also be used to identify and quantifyimmune complexes. Sandwich assays are a mirror image of non-competitiveELISAs in that antibodies are bound to the solid phase and antigen inthe biological sample is measured. These assays are particularly usefulin detecting antigens, having multiple epitopes, that are present at lowconcentrations. This technique requires excess antibody to be attachedto a solid phase, such as the biochip. The bound antibody is thenincubated with the biological samples, and the antigens in the sampleare allowed to form immune complexes with the bound antibody. The immunecomplex is incubated with an enzyme-linked secondary antibody, whichrecognizes the same or a different epitope on the antigen as the primaryantibody. Hence, enzyme activity is directly proportional to the amountof antigen in the biological sample. D. M. Kemeny & S. J. Challacombe,ELISA and Other Solid Phase Immunoassays (1988).

Typical enzymes that can be linked to secondary antibodies includehorseradish peroxidase, glucose oxidase, glucose-6-phosphatedehydrogenase, alkaline phosphatase, β-galactosidase, and urease.Secondary antigen-specific antibodies linked to various enzymes arecommercially available from, for example, Sigma Chemical Co. andAmersham Life Sciences (Arlington Heights, Ill.).

Competitive ELISAs are similar to noncompetitive ELISAs except thatenzyme linked antibodies compete with unlabeled antibodies in thebiological sample for limited antigen binding sites. Briefly, a limitednumber of antigens are bound to the solid support. Biological sample andenzyme-labeled antibodies are added to the solid support.Antigen-specific antibodies in the biological sample compete withenzyme-labeled antibodies for the limited number of antigens bound tothe solid support. After immune complexes have formed, nonspecificallybound antibodies are removed by washing, enzyme substrate is added, andthe enzyme activity is measured. No secondary antibody is required.Because the assay is competitive, enzyme activity is inverselyproportional to the amount of antibodies in the biological sample.

An alternative competitive ELISA can also be used within the scope ofthe present invention. In this alternative embodiment, limited amountsof antibodies from the biological sample are bound to the surface of thesolid support as described herein. Labeled and unlabeled antigens arethen brought into contact with the solids support such that the labeledand unlabeled antigens compete with each other for binding to theantibodies on the surface of the solid support. After immune complexeshave formed, nonspecifically bound antigens are removed by washing. Theimmune complexes are detected by incubation with an enzyme-linkedsecondary antibody, which recognizes the same or a different epitope onthe antigen as the primary antibody, as described above. The activity ofthe enzyme is then assayed, which yields a signal that is inverselyproportional to the amount of antigen present.

Homogeneous immunoassays can also be used when practicing the method ofthe present invention. Homogeneous immunoassays may be preferred fordetection of low molecular weight compounds, such as hormones,therapeutic drugs, and illegal drugs that cannot be analyzed by othermethods, or compounds found in high concentration. Homogeneous assaysare particularly useful because no separation step is necessary. R. C.Boguslaski et al., Clinical Immunochemistry: Principles of Methods andApplications (1984).

In homogeneous techniques, bound or unbound antigens are enzyme-linked.When antibodies in the biological sample bind to the enzyme-linkedantigen, steric hindrances inactivate the enzyme. This results in ameasurable loss in enzyme activity. Free antigens (i.e., notenzyme-linked) compete with the enzyme-linked antigen for limitedantibody binding sites. Thus, enzyme activity is directly proportionalto the concentration of antigen in the biological sample.

Enzymes useful in homogeneous immunoassays include lysozyme,neuramimidase, trypsin, papain, bromelain, glucose-6-phosphatedehydrogenase, and β-galactosidase. T. Persoon, Immunochemical Assays inthe Clinical Laboratory, 5 Clinical Laboratory Science 31 (1992).Enzyme-linked antigens are commercially available or can be linked usingvarious chemicals well known in the art, including glutaraldehyde andmaleimide derivatives.

Prior antibody profiling technology involves an alkaline phosphataselabeled secondary antibody with 5-bromo-4-chloro-3′-indolylphosphatep-toluidine salt (BCIP) and nitro-blue tetrazolium chloride (NBT), bothof which are commercially available from a variety of sources, such asfrom Pierce Chemical Co. (Rockford, Ill.). The enzymatic reaction formsan insoluble colored product that is deposited on the surface of themembrane strips to form bands wherever antigen-antibody complexes occur.This method is suboptimal in a biochip format since it is difficult toquantify and since colorimetric methods are typically less sensitivethan assays base on fluorescence or luminescence.

Fluorescent immunoassays can also be used when practicing the method ofthe present invention. Fluorescent immunoassays are similar to ELISAsexcept the enzyme is substituted for fluorescent compounds calledfluorophores or fluorochromes. These compounds have the ability toabsorb energy from incident light and emit the energy as light of alonger wavelength and lower energy. Fluorescein and rhodamine, usuallyin the form of isothiocyanates that can be readily coupled to antigensand antibodies, are most commonly used in the art. D. P. Stites et al.,Basic and Clinical Immunology (1994). Fluorescein absorbs light of 490to 495 nm in wavelength and emits light at 520 nm in wavelength.Tetramethylrhodamine absorbs light of 550 nm in wavelength and emitslight of 580 nm in wavelength. Illustrative fluorescence-based detectionmethods include ELF-97 alkaline phosphatase substrate (Molecular ProbesInc., Eugene, Oreg.); PBXL-1 and PBXL-3 (phycobilisomes conjugated tostreptavidin) (Martek Biosciences Corp., Columbia, Md.); FITC and TexasRed labeled goat anti-human IgG (Jackson ImmunoResearch Laboratories,Inc., West Grove, Pa.); and B-Phycoerythrin and R-Phycoerythrinconjugated to streptavidin (Molecular Probes Inc.). ELF-97 is anonfluorescent chemical that is digested by alkaline phosphatase to forma fluorescent molecule. Because of turn over of the alkalinephosphatase, use of the ELF-97 substrate results in signalamplification. Fluorescent molecules attached to secondary antibodies donot exhibit this amplification.

Phycobiliproteins isolated from algae, porphyrins, and chlorophylls,which all fluoresce at about 600 nm, are also being used in the art. I.Hemmila, Fluoroimmunoassays and Immunofluorometric Assays, 31 Clin.Chem. 359 (1985); U.S. Pat. No. 4,542,104. Phycobiliproteins andderivatives thereof are commercially available under the namesR-phycoerythrin (PE) and Quantum Red™ from, for example, Sigma ChemicalCo.

In addition, Cy-conjugated secondary antibodies and antigens are usefulin immunoassays and are commercially available. Cy-3, for example, ismaximally excited at 554 nm and emits light of between 568 and 574 nm.Cy-3 is more hydrophilic than other fluorophores and thus has less of atendency to bind nonspecifically or aggregate. Cy-conjugated compoundsare commercially available from Amersham Life Sciences.

Illustrative luminescence-based detection methods include CSPD and CDPstar alkaline phosphatase substrates (Roche Molecular Biochemicals); andSuperSignal® horseradish peroxidase substrate (Pierce Chemical Co.,Rockford, Ill.).

Chemiluminescence, electroluminescence, and electrochemiluminescence(ECL) detection methods are also attractive means for quantifyingantigens and antibodies in a biological sample. Luminescent compoundshave the ability to absorb energy, which is released in the form ofvisible light upon excitation. In chemiluminescence, the excitationsource is a chemical reaction; in electroluminescence the excitationsource is an electric field; and in ECL an electric field induces aluminescent chemical reaction.

Molecules used with ECL detection methods generally comprise an organicligand and a transition metal. The organic ligand forms a chelate withone or more transition metal atoms forming an organometallic complex.Various organometallic and transition metal-organic ligand complexeshave been used as ECL labels for detecting and quantifying analytes inbiological samples. Due to their thermal, chemical, and photochemicalstability, their intense emissions and long emission lifetimes,ruthenium, osmium, rhenium, iridium, and rhodium transition metals arefavored in the art. The types of organic ligands are numerous andinclude anthracene and polypyridyl molecules and heterocyclic organiccompounds. For example, bipyridyl, bipyrazyl, terpyridyl, andphenanthrolyl, and derivatives thereof, are common organic ligands inthe art. A common organometallic complex used in the art includestris-bipyridine ruthenium (II), commercially available from IGEN, Inc.(Rockville, Md.) and Sigma Chemical Co.

Advantageously, ECL can be performed under aqueous conditions and underphysiological pH, thus minimizing biological sample handling. J. K.Leland et al., Electrogenerated Chemiluminescence: AnOxidative-Reduction Type ECL Reactions Sequence Using Triprophyl Amine,137 J. Electrochemical Soc. 3127-3131 (1990); WO 90/05296; U.S. Pat. No.5,541,113. Moreover, the luminescence of these compounds may be enhancedby the addition of various cofactors, such as amines.

In practice, a tris-bipyridine ruthenium (II) complex, for example, maybe attached to a secondary antibody using strategies well known in theart, including attachment to lysine amino groups, cysteine sulfhydrylgroups, and histidine imidazole groups. In a typical ELISA immunoassay,secondary antibodies would recognize ISAs bound to antigens, but notunbound antigens. After washing nonspecific binding complexes, thetris-bipyridine ruthenium (II) complex would be excited by chemical,photochemical, and electrochemical excitation means, such as by applyingcurrent to the biochip. E.g., WO 86/02734. The excitation would resultin a double oxidation reaction of the tris-bipyridine ruthenium (II)complex, resulting in luminescence that could be detected by, forexample, a photomultiplier tube. Instruments for detecting luminescenceare well known in the art and are commercially available, for example,from IGEN, Inc.

Solid state color detection circuitry can also be used to monitor thecolor reactions on the biochip and, on command, compare the colorpatterns before and after the sample application. A color camera imagecan also be used and the pixel information analyzed to obtain the sameinformation.

Still another method involves detection using a surface plasmonresonance (SPR) chip. The surface of the chip is scanned before andafter sample application and a comparison is made. The SPR chip relieson the refraction of light when the molecules of interest are exposed toa light source. Each molecule has its own refraction index by which itcan be identified. This method requires precise positioning and controlcircuitry to scan the chip accurately.

Yet another method involves a fluid rinse of the biochip with afluorescing reagent. The microlocations that combine with the biologicalsample will fluoresce and can be detected with a charge-coupled device(CCD) array. The output of such a CCD array is analyzed to determine theunique pattern associated with each sample. This approach avoids theproblems associated with scanning technologies. Speed is not a factorwith any of the methods since the chemical combining of sample andreference takes minutes to occur.

Moreover, array scanners are commercially available, such as fromGenetic MicroSystems. The GMS 418 Array Scanner uses laser optics torapidly move a focused beam of light over the biochip. This system usesa dual-wavelength system including high-powered, solid-state lasers thatgenerate high excitation energy to allow for reduced excitation time. Ata scanning speed of 30 Hz, the GMS 418 can scan a 22×75-mm slide with10-μm resolution in about 4 minutes.

Software for image analysis obtained with an array scanner is readilyavailable. Available software packages include ImaGene (BioDiscovery,Los Angeles, Calif.); ScanAlyze (available at no charge; developed byMike Eisen, Stanford University); De-Array (developed by Yidong Chen andJeff Trent of the National Institutes of Health; used with IP Lab fromScanalytics, Fairfax, Va.); Pathways (Research Genetics, Huntsville,Ala.); GEM tools (Incyte Pharmaceuticals, Inc., Palo Alto, Calif.); andImaging Research (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.).

Once interactions between the antigens and ISAs have been identified andquantified, the signals may be digitized. The digitized antibody profileserves as a signature that identifies the source of the biologicalsample. Depending on the biochip used, the digitized data may takenumerous forms. For example, the biochip may comprise an array with 10columns and 10 rows for a total number of 100 microlocations. Eachmicrolocation contains at least one antigen. After the biological samplecontaining the ISAs is added to each microlocation and allowed toincubate, interactions between antigens and ISAs in the biologicalsample are identified and quantified. In each microlocation, aninteraction between the antigen at that microlocation and the ISAs inthe biological sample either do or do not result in a quantifiablesignal. In one preferred embodiment, the results of the antibody profileare digitized by ascribing each one of the 100 microlocations anumerical value of either “0,” if a quantifiable signal was notobtained, or “1,” if a quantifiable signal was obtained. Using thismethod, the digitized antibody profile comprises a unique set of 0's and1's.

The numerical values “0” or “1” will, of course, preferably benormalized to signals obtained in internal control microlocations sothat digitized antibody profiles obtained at a later time can beproperly compared. For example, one or several of the microlocationswill contain a known antigen, which will remain constant over time.Therefore, if subsequent biological sample is more or less dilute than aprevious biological sample, the signals can be normalized using thesignals from the known antigen.

It will be appreciated by one skilled in the art that other methods ofdigitizing the antibody profile exist and may be used. For example,rather than ascribing each microlocation with a numerical value of “0”or “1,” the numerical value may be incremental and directly proportionalto the strength of the signal.

By digitizing the antibody profile signals, the biochemical results canbe entered into a computer and quickly accessed and referenced. Withinseconds of having the antibody profile digitized, a computer can comparea previously digitized antibody profile to determine whether there is amatch. If a matching antibody profile is in the database, a positiveidentification of the source of the biological sample can be made. Thus,the method of the present invention can both discriminate and positivelyidentify the source of a biological sample.

In a preferred embodiment of the invention, the present method is usedfor forensic analysis for matching a biological sample to a criminalsuspect. Forensic samples obtained from crime scenes are often subjectto drying of the samples, small sample sizes, mixing with samples frommore than one individual, adulteration with chemicals, and the like. Thepresent method provides the advantages of rapid analysis, simplicity,low cost, and accuracy for matching forensic samples with suspects. Forexample, the forensic sample and a sample from one or more suspects areobtained according to methods well known in the art. Antibody profilesfor each of the samples are prepared, as described herein. The antibodyprofiles are then compared. A match of antibody profiles means that theforensic sample was obtained from the matching suspect. If no match ofantibody profiles is obtained, then none of the suspects was the sourceof the forensic sample.

In another preferred embodiment of the invention, the present method isused for drug testing of individuals. For example, in many work placesit is a condition of obtaining or maintaining employment to be free ofillegal drug use. The presence of illegal drugs in the bloodstream of aperson can be detected by the present method by antibody capture orsimilar methods. Moreover, as described in WO 97/29206, the drug testand the identity of the sample can be correlated in a single test. Drugtests are also important in certain animals, such as horses and dogsinvolved in racing.

EXAMPLE 1

The law enforcement community has demonstrated several needs associatedwith drug testing of suspects including dealing with privacy issuesassociated with sample collection, maintenance of sample chain ofcustody, prevention of sample adulteration by the suspect, andfacilitating more rapid turn around time on sample analyses. Currentdrug testing protocols utilize urine samples and, occasionally, bloodsamples. Invasion of privacy is a continuing problem with urine samplessince it is necessary to observe the individual providing the sample tomaintain the chain of custody and eliminate the possibility of sampleswitching or adulteration. Urine samples are also not a good indicatorof the current level of intoxication since many drug metabolitescontinue to be excreted into urine for days or weeks after the drugs areinitially taken. While blood samples do not suffer from these problems,collecting blood is an invasive procedure requiring special facilitiesand trained personnel that may not always be available when the needarises. It is necessary for law enforcement personnel to maintain strictchain of custody for all samples collected to ensure that mishandling ordeliberate tampering do not occur. A break or even a perceived break inthe chain of custody can result in evidence being dismissed outright orgiven little weight.

The present invention solves these issues in several ways. First,incorporation of the antibody profiling identification assay into thedrug test makes identification of the sample donor integral to the testand eliminates the need for complex chain of custody procedures. Second,a saliva-based drug test is better than a urine test because drug levelsin saliva can be readily correlated with drug levels in blood (W.Schramm et al., Drugs of Abuse in Saliva: A Review, 16 J. Anal.Toxicology 1-9 (1992); E. J. Cone, Saliva Testing for Drugs of Abuse,694 Ann. N.Y. Acad. Sci. 91-127 (1995)), therefore providing a betterindicator of current drug use (D. A. Kidwell et al., Testing for drugsof abuse in saliva and sweat, 713 J. Chrom. B 111-135 (1998)). Salivasamples from a suspect can also be collected easily in view of a lawenforcement officer without invasion of privacy or with invasivemethods. Finally, the present test is easy to use and can be quicklyperformed by law enforcement personnel on site, instead of requiring thedays to weeks necessary at distant centralized laboratories. V. S.Thompson et al., Antibody profiling as an identification tool forforensic samples, 3576 Investigation and Forensic Science Technologies52-59 (1999).

In this example, an antibody-based test is provided for two commonillicit drugs (cocaine and methamphetamine). These drugs are among themost commonly abused, and their use is on the rise. S. B. Karch, DrugAbuse Handbook (CRC Press, 1998); L. D. Bowers, Athletic Drug Testing,17 Sports Pharmacology 299-318 (1998).

Materials and Methods. Goat anti-rabbit IgG antibodies conjugated toalkaline phosphatase were obtained from Jackson ImmunoResearch (WestGrove, Pa.). Rabbit anti-human IgA antibodies were purchased from U.S.Biological (Swampscott, Mass.). Seablock™, nitro-blue tetrazoliumchloride/5-bromo-4-chloro-3′-indolylphosphate p-toluidine salt(NBT/BCIP), p-nitrophenyl phosphate disodium salt (PNPP), EZ-Link™maleimide activated alkaline phosphatase kits, and FreeZyme® conjugatepurification kits were obtained from Pierce Chemical (Rockford, Ill.).Monoclonal antibodies against benzoylecgonine and methamphetamine, andbovine serum albumin (BSA) conjugates of methamphetamine andbenzoylecgonine were purchased from O.E.M Concepts (Toms River, N.J.).Cocaine and methamphetamine hydrochloride salts were obtained fromSigma-Aldrich (St. Louis, Mo.). Antibody Profiling strips were purchasedfrom Miragen, Inc. (Irvine, Calif.). Strips used for the combineddrug-AbP test were produced according to the protocol of A. M.Francoeur, Antibody fingerprinting: a novel method for identifyingindividual people and animals, 6 Bio/Technology 822-825 (1988). Salivasamplers from Saliva Diagnostic Systems (Vancouver, Wash.), Ora SureTechnologies, Inc. (Bethlehem, Pa.), and Sarstedt, Inc. (Newton, N.C.)were used to collect saliva samples from volunteers.

A saliva-based AbP assay was developed through modification of anearlier protocol designed for processing blood samples. T. F. Unger & A.Strauss, Individual-specific antibody profiles as a mean of newborninfant identification, 15 J. Perinatology 152-155 (1995). Briefly, 500μl of saliva sample diluted with 1.0 ml of PBST (50 mM phosphatebuffered saline, 0.2% Tween 20) was incubated with an AbP stripovernight for a minimum of 16 hours, and excess sample was washed offwith PBST. Next, the strip was incubated successively with 100 ng/mlrabbit anti-human IgA for 1 hour and 100 ng/ml goat anti-rabbitIgG-alkaline phosphatase conjugate for 30 minutes with wash steps inbetween incubations. The strip was washed again with PBST and aprecipitation substrate for alkaline phosphatase, NBT/BCIP, was added toallow development of bands on the strip.

The Saliva Sampler™ (Saliva Diagnostic Systems) and the Salivette™(Sarstedt, Inc.) saliva collection systems were examined forcompatibility with the AbP assay. The Saliva Sampler™ system comprises acotton pad attached to a plastic handle. A window in the handle turnsblue when sufficient sample has been collected. The pad is placed in apreservative buffer after collection. The Salivette™ is a cotton rollplaced in the mouth for about 10 minutes and then centrifuged in aplastic tube to collect sample. Both types of samplers were placed inthe gingival crevice of the mouth for sample collection. The quality ofsamples as a function of storage time at temperatures of −20° C., 4° C.,and 25° C. was assessed by performing AbP on samples collected with bothsamplers.

Five volunteers participated in studies to compare blood AbP patternswith those obtained from saliva samples. Protocols for use of humansubjects were conducted in accordance with the Idaho NationalEngineering and Environmental Laboratory Institutional Review Board.Blood samples were collected in tubes containing the anticoagulant EDTAand were used immediately. Saliva was collected using the SalivaSampler™ saliva collection system. Paired blood and saliva samples wereanalyzed using the blood protocol of Unger & Strauss, supra, and thesaliva AbP test described above.

Four additional volunteers participated in a saliva adulteration studyto assess the effects of various foods and beverages on the AbP assay.The volunteers were given butterscotch and lemon hard candy, sugar andsugar-free gum, sugar and sugar-free cola, and milk chocolate. Aftereating the above, they were asked to collect saliva samples using theprovided saliva samplers. Volunteers were also asked to consume alcohol,drink coffee, eat a food of their choice, and brush their teeth prior togiving samples. A volunteer who was a smoker provided a sample aftersmoking a cigarette. Baseline samples were also collected from thevolunteers.

Monoclonal antibodies against methamphetamine and benzoylecgonine wereconjugated to alkaline phosphatase using the Pierce EZ-Link™ maleimideactivated alkaline phosphatase kit according to the manufacturer'sprotocols. Unconjugated antibody was separated from the antibody-enzymeconjugate using the FreeZyme™ conjugate purification kit according tothe manufacturer's protocols.

Competitive enzyme linked immunosorbent assays (ELISAs) were developedfor both cocaine and methamphetamine. The BSA conjugates ofmethamphetamine or benzoylecgonine were diluted in 50 mM carbonatebuffer, pH 9.6, and 50 μl was added to each well of a 96-well microtiterplate. The plate was incubated overnight at 4° C. to allow theconjugates to bind to the well surfaces. The plate was then washed withPBST to remove excess BSA conjugate. Next, 50 μl of either cocaine ormethamphetamine solution in the concentration range from 0 to 1000 μg/mlwas added to the plate and 50 μl of either monoclonalanti-benzoylecgonine or anti-methamphetamine conjugated with alkalinephosphatase was added. During this step, the immobilized BSA drugconjugate competed with the free drug in solution for binding sites onthe antibodies. After the competition reaction was complete, the unboundantibodies and free drug were washed away. Finally, 100 μl of solublealkaline phosphatase substrate (PNPP) solution was added to the wells toreact with the alkaline phosphatase bound to the well surfaces throughthe anti-drug antibodies. The reaction was stopped after 20-30 minutesby addition of 25 μl of 3 M NaOH, and the absorbance of each well wasread at 405 nm using a Tecan Spectra microplate reader.

Polyvinylidene fluoride (PVDF) membrane is used in the manufacture ofthe Miragen AbP strips, and was used to assess the feasibility ofbinding the cocaine- and methamphetamine-BSA conjugates to the itssurface. The PVDF membrane was cut into strips the same size as thoseused in the AbP assay. Four strips were prepared for each drug and 10 μlspots of either drug-BSA conjugate were placed at three locations oneach strip for analysis in triplicate. The strips were dried at 35° C.for one hour prior to use. Non-specific binding sites on the strips wereblocked with PBST containing 1 mg/mL BSA for one hour and then rinsedwith PBST. Cocaine and methamphetamine solutions were prepared in PBSTat concentrations of 0, 0.1, 10, and 1000 μg/ml. Next, 750 μl of cocaineor methamphetamine solution were added to the strips and another 750 μlof anti-benzoylecgonine or anti-methamphetamine antibodies conjugatedwith alkaline phosphatase were added and allowed to incubate for onehour. During this time a competitive reaction between the free andimmobilized drug for antibody binding sites took place. The strips werewashed to remove unbound antibodies and drugs and the NBT/BCIP substratewas added. The strips were allowed to develop for 15 minutes.

A combined AbP-drug assay was prepared by placing 10 μl spots of bothmethamphetamine and benzoylecgonine-BSA conjugate onto the blank bottomportion of the AbP strip and allowing them to dry for one hour at 35° C.Saliva samples from three individuals were collected using Ora Suresamplers. Half of the saliva sample was spiked with 1000 μg/ml ofcocaine or methamphetamine. The strips were blocked with PBST containing1.0 mg/ml BSA for one hour and rinsed with PBST. Next, 500 μl of spikedor unspiked saliva was added to the strips along with alkalinephosphatase conjugated anti-benzoylecgonine and anti-methamphetamineantibodies and allowed to incubate over night at room temperature. Thestrips were washed with PBST and the AbP assay was conducted asdescribed above.

Results and Discussion. The saliva-based AbP assay was optimized throughvariation of reagent concentrations, sample volumes, and incubationtimes. Illustrative results of antibody profiles obtained from salivasamples are shown in FIG. 1. Compared to the blood-based AbP assay, thesaliva assay takes much longer (18 hours versus 2 hours) and requires a10-fold larger amount of sample. This is due to the 100-fold lowerlevels of total antibody present in saliva as compared to blood. Parry,Tests for HIV and hepatitis viruses, 694 Annals N.Y. Acad. Sci. 221(1993).

The stability of antibodies present in the saliva samples collectedusing the Saliva Sampler™ or the Salivette™ systems was determined bystorage at −20° C., 4° C., and 25° C. and AbP testing of samples dailyover the period of one week to see if there were any changes in thepatterns observed. Fresh saliva samples from either sampler gave thebest results. The stability over time of samples collected with theSaliva Sampler™ system was superior to samples collected with theSalivette™ system at all temperatures. The preservative storage bufferprovided with the Saliva Sampler™ system appears to prevent antibodydegradation due to bacterial contamination, while the Salivette™ samplerincludes no preservative.

The samples collected with the Saliva Sampler™ system and maintained atroom temperature showed no change in pattern over a five-day period.This result is in contrast to the results obtained with samples storedin a refrigerator, which showed marked deterioration even after a fewhours of storage. It is not clear why this occurred. Frozen samples alsoshowed some deterioration due to damage caused by freeze-thaw cycles,but prolonged storage at freezing temperatures resulted in no furtherdegradation. Since Saliva Sampler™ saliva collection systems hadsuperior storage properties and were easier to use, they were used forthe adulteration studies.

Blood AbP patterns were compared to saliva AbP patterns to determine ifthe ISAs present in those samples were the same. The results showed thatthe patterns obtained from the two different samples differed markedly(FIG. 2). This result was somewhat surprising since saliva is a filtrateof blood, and it was expected that the ISAs present in saliva would bethe same as those present in blood. The different patterns probablyresulted from the isotype of antibody examined in each case. In bloodIgG antibodies were analyzed since they are the most prevalent. Insaliva, IgA antibodies are more prevalent and were analyzed. After theabove result was obtained, saliva samples were also analyzed for IgGantibodies to determine if those patterns would be the same as thosefrom the blood patterns. However, this was unsuccessful due to theextremely low levels of IgG antibodies present in saliva.

The saliva adulteration studies showed that virtually no changesoccurred in the antibody profiles when any of the adulterants werepresent (FIG. 3). In some cases a band might be darker or lighter, butthere appeared to be no missing or additional bands present. Since thiswas a preliminary study, the adulterants examined were easily obtainableitems that might be used during the course of ordinary life. However, asa quick search of the Internet reveals, there are many proposed methodsto beat urine-based drug tests including ingestion of and adulterationof samples with various substances that are being sold by these sites.The adulteration results shown here are promising since it appears thatthe AbP test is not affected by foods that may be commonly consumedbefore taking a saliva test.

Immunoassay tests for both cocaine and methamphetamine were developedusing a direct competitive assay. An anti-benzoylecgonine antibody wasused for the cocaine assay; however, this antibody gave the sameresponse to cocaine as to benzoylecgonine (the primary metabolite ofcocaine) so it did not effect the results of the assay. In this assay,drug present in a sample competes for binding sites on enzyme labeledantibodies with a BSA-conjugated drug immobilized to the surface of awell of a microtiter plate. In samples with large drug concentrations,most of the antibody-enzyme conjugate will bind to the drug in solutionand will be washed away during the final step. Therefore, there will bevery little enzyme present in the microtiter plate and the amount ofcolor development will be low. Conversely, if there is no drug in thesample, the antibodies will bind to the immobilized drugs and stay inthe wells after the wash step resulting in strong color development.This results in a signal that is inversely proportional to the drugconcentration (FIGS. 4 and 5). The linear range for cocaine detectionwas from 0.1 to 5 μg/ml and for methamphetamine was from 0.1 to 10μg/ml. This range covers the cutoff values for these drugs (0.3 and 1.0μg/ml, respectively) currently set by the Substance Abuse and MentalHealth Services Administration. M. Peat & A. E. Davis, Drug AbuseHandbook (CRC Press, Boca Raton, Fla. 1998).

Using the optimum concentrations of BSA-drug conjugates determinedduring the ELISA studies, the drug assays were conducted on the PVDFmembranes. Because of the inverse relationship of the immunoassay todrug concentration, a dark spot was observed when the concentration ofdrugs was low, and spots gradually disappeared as the drug concentrationincreased (FIGS. 6 and 7).

Since the drug test on the PVDF membranes were promising, thefeasibility of combining the two drug tests with the AbP assay wasassessed. Antibody profile patterns from the three individuals did notchange regardless of whether the drug was present or not (FIG. 8). Thisresults shows that the presence of the drugs did not interfere with thereagents used to perform the antibody profiling assay.

EXAMPLE 2

In this example the procedure of Example 1 is followed except thatfractionated HeLa cell antigens are immobilized on a PVDF membrane in apredetermined pattern as a two-dimensional array. Additionally, cocaineand methamphetamine are immobilized on the membrane as additional spotson the array. After development of color as described, results aresubstantially similar to those of Example 1.

EXAMPLE 3

In this example the procedure of Example 2 is followed except that thearray is immobilized on a glass slide.

1. A method for analyzing biological material comprising individual-specific antibodies, the method comprising: forming an array comprising multiple antigens attached to a surface of a solid support in a preselected pattern such that locations of the multiple antigens are known; obtaining a sample of a biological material having individual-specific antibodies and contacting the array with the sample to bind at least a portion of the individual-specific antibodies to the multiple antigens of the array, to form immune complexes; washing the array containing the immune complexes; detecting the immune complexes; and identifying the immune complexes on the array, to obtain an antibody profile.
 2. The method of claim 1, wherein forming an array comprises attaching the multiple antigens to the solid support through a covalent bond.
 3. The method of claim 1, comprising obtaining a sample of a biological material selected from the group of biological material consisting of tissue, blood, saliva, urine, perspiration, tears, semen, serum, plasma, amniotic fluid, pleural fluid, cerebrospinal fluid, and combinations thereof.
 4. The method of claim 1, wherein forming the array comprises attaching multiple antigens to a solid support composed of glass or silica.
 5. The method of claim 1, wherein detecting the immune complexes comprises treating the array such that the presence of immune complexes at a location is characterized by a color change at the location.
 6. The method of claim 5, wherein detecting the immune complexes comprises obtaining an output using a charge-coupled device and wherein the color change comprises fluorescence or luminescence emission.
 7. The method of claim 1, wherein detecting the immune complexes further comprises monitoring the array with solid state color detection circuitry and comparing color patterns before and after detecting the immune complexes.
 8. The method of claim 1, wherein detecting the immune complexes further comprises obtaining a color camera image before contacting the array with the sample and after detecting the immune complexes, and analyzing pixel information obtained therefrom.
 9. The method of claim 1, wherein detecting the immune complexes further comprises scanning the array before and after contacting the array with the sample, wherein the solid support is a surface plasmon resonance chip.
 10. The method of claim 1, wherein forming the array comprises attaching a first subset of antigens configured for obtaining an antibody profile and a second subset of at least one antigen configured for assaying for a selected analyte in the sample.
 11. The method of claim 10, wherein attaching the second subset of at least one antigen comprises attaching at least one drug.
 12. The method of claim 11, wherein attaching at least one drug comprises attaching a drug selected from the group consisting of marijuana, cocaine, methamphetamine, amphetamine, heroin, methyltestosterone, mesterolone and combinations thereof.
 13. The method of claim 2, wherein obtaining a sample of a biological material comprises obtaining the biological material from a forensic sample.
 14. The method of claim 13, further comprising comparing the antibody profile obtained from the biological material from the forensic sample to an antibody profile prepared from a biological sample obtained from a suspect. 